Programmed DNA rearrangements are important developmental processes that occur in a wide variety of organisms, and are relevant to the understanding of many human diseases including cancer. This proposal will examine the molecular mechanisms of three such processes in the model eukaryote Tetrahymena thermophila. They are: DNA deletion, chromosome breakage and gene amplification. These processes are precisely regulated, and can be induced to occur synchronously in large populations. Past studies have revealed interesting nucleotide sequence features that are responsible for the regulation of these processes. In this proposal a multi prone approach will be taken to analyze their cis-acting sequences and trans-acting factors, relying partly on a DNA transformation method developed previously in this laboratory. Chromosome breakage is an event that cleaves DNA and adds new telomeric sequences to the free ends. It occurs at 50-200 specific genomic sites that contain a 15 bp sequence known to be the signal for this process. In this proposal attempts will be made to establish an in vitro reaction system to analyze the details of the reaction steps, and to identify proteins or other macromolecules involved. In addition, a special transgenic strain will be created and used to screen for mutants defective in this process. Analysis of these mutants will help us understand its regulation and biological roles. DNA deletion is a complex process that occurs at several thousand genomic locations. The deleted DNAs are diverse in size and sequence, and together comprise about 15% of the genome. Studies of one such deletion element have revealed two types of essential cis-acting sequences: a pair of flanking regulatory sequences and a set of internal activating sequences. Studies are now proposed to define these controlling sequences, to find out how they specify the deletion site, and to learn if the same rule applies to other deletion elements. An in vitro reaction system will also be set up to identify reaction intermediates and isolate trans-acting factors. In addition, efforts will be made to characterize proteins that are specifically expressed at this stage of development. One such protein has been purified recently and show interesting properties implying its involvement in this process. Finally, amplification of the ribosomal RNA gene will be analyzed by dissecting its cis-acting sequences. This gene is excised from the chromosome and become highly amplified during development, which is propagated as free molecules during vegetative growth. A special transformation vector has been developed and will be used to define the minimal sequence required for the replication of this molecule during growth, and uncover other sequences involved in its amplification during development. These studies should give new insights into these intriguing processes and reveal their regulatory mechanisms. They may also help us understand their possible relationships to other cellular processes, such as chromosome condensation, mitosis or meiosis, which never occur in these nuclei after DNA rearrangements.